Vascularised tissue graft

ABSTRACT

A method of producing vascularised tissue utilizing a vascular pedicle enclosed in a chamber and implanted in a donor is provided. A vascularised tissue graft suitable for transplantation is also provided. The invention also encompasses a method of repairing a tissue deficit using a vascularised tissue graft.

[0001] This invention relates to the fields of tissue engineering andtransplantation, and particularly to the generation of vascularisedtissue.

BACKGROUND OF THE INVENTION

[0002] Tissue engineering utilising homologous starting material offersthe prospect of replacing missing or non-functioning body parts withnewly created, living tissue. It has the potential to minimise loss oftissue and resultant pain from the donor site experienced inconventional reconstructive surgery or to recreate specialized tissuefor which there is no donor site, while obviating the long-termimmunosuppression required for heterologous transplantation.

[0003] It combines the techniques of tissue culture, the creation ofbio-compatible materials and the manipulation of angiogenesis in orderto create new, vascularised tissue to replace damaged tissue or tissuewhich is congenitally absent.

[0004] One of the major challenges faced in tissue engineering is tocreate differentiated tissue of the appropriate size and shape. Tissuecreated without a functional vasculature is strictly limited in size bythe constraints of oxygen diffusion; if the tissue is too large it willbecome necrotic before the host has time to create a new blood vesselsupply. Thus there are many advantages in creating new tissue containinga functional vasculature. Additionally, as the new tissue may need to beproduced at a site on the body remote from the defect, or on animmunosuppressed carrier animal or in vitro with an extracorporealcirculation, the blood supply for the new tissue must be defined, sothat it can be brought with the tissue intact to the site ofreconstruction.

[0005] The creation of skin flaps, a living composite of skin and itsunderlying fat, is a common technique used to repair tissue defects inreconstructive surgery. Because these flaps must retain their bloodsupply to remain viable after transplantation, the origin of the flapsis limited to those areas where there is an anatomically recognisedblood vessel source. In order to overcome this limitation, skin flapscan be “pre-fabricated” by implanting short segments of blood vesselsinto a desired site, and utilising the resultant angiogenesis tovascularise a flap of the desired size and composition. Subsequentlythis vascularised flap can be transferred by microsurgery to the regionof interest. This technique is, however, limited by the availability ofdonor tissue, and the disfigurement that results at the donor site.

[0006] In an extension to this technique, Erol and Spira (1980)demonstrated that the creation of an anastomosed arterio-venous (AV)loop beneath a skin graft could produce a vascularised skin flap.

[0007] However, while the generation of vascularised skin using an AVloop has been demonstrated, the production of other vascularised tissuessuitable for grafting remains elusive. Vascularised adipose tissue, forexample, is often demanded in reconstructive procedures; however, donormature adipose tissue is extremely fragile, and will rapidly becomenecrotic if not immediately re-connected to a functional blood supply.Furthermore, the use of conventional autologous transplantationtechniques involves “robbing Peter to pay Paul”, producing disfigurementat the donor site. The ability to produce new tissue with a definedvasculature would overcome this major shortcoming.

[0008] Khouri et al. (1993) and Tanaka et al. (1996) have demonstratedthat an arteriovenous loop could intrinsically generate new,vascularised tissue when it was lifted from the body, sandwiched betweensheets of collagenous matrix and isolated from the surrounding tissuewithin a plastic chamber. In the model described by Khouri et al., thegeneration of new tissue relied on the addition of recombinantBB-homodimer of Platelet-Derived Growth Factor (BB-PDGF), and even withthis supplement the tissue was labile, peaking in volume at 15 days andsubsiding by 30 days. Similarly, tissue growth in Tanaka's model, wherethe chamber was supplemented with β-Fibroblast Growth Factor (β-FGF orFGF-2), continued to increase in volume, peaking at 2 weeks, butreturned to the levels of the unsupplemented control chambers after 4weeks. This AV loop model is not generally known in th field of tissueengineering.

[0009] The classical notion that mature tissues do not contain stemcells has changed considerably in recent years. Many mature tissueswhich were previously regarded as largely non-self renewing are nowconsidered to harbour a stem cell population. These stem cells possessthe potential to change their phenotype in response to theirenvironment, and may be able to provide a self-replenishing stem cellpopulation (Prockop, 1997). Micro-environmental cues are considered toplay a significant role in determining the behaviour of stem cells, forexample, in initiating stem cell division and differentiation and/ormaintaining stem cell quiescence. The cues and mechanisms behind theseprocesses are far from being understood. However, it is clear that theability to recruit, stimulate, proliferate and differentiate stem cellsis the crux of tissue engineering. The behaviour of stem cells islargely studied in vitro, although a small number of in vivo studieshave examined the behaviour of stem cells when injected either under thecapsule of mature organs or systemically. These studies have a number oflimitations in furthering the knowledge of the use of stem cells fortissue engineering. In particular, when the stem cells are injected intomature organs they must interact with an established micro-environmentand derive a limited neovasculature from the host organ; when they aresystemically injected they become widely dispersed. In order for stemcells to generate organs, it is expected that they will require anexpandable vascular supply to accommodate and service de novo tissuegeneration. In order to assist in directing stem cell expansion,development and differentiation, an expandable microenvironmentcomprising an inert support and/or extracellular matrix is also expectedto be required. We have now developed a model which satisfies theserequirements, and holds great promise for the study of stem cells. Itsapplication to tissue engineering is a significant advance in the stateof the art.

[0010] It will be clearly understood that, although a number of priorart publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

[0011] We have now developed a system for producing vascularised grafttissue, which is useful in transplant and reconstructive surgery, andalso provides a useful model system.

SUMMARY OF THE INVENTION

[0012] In a first aspect, the invention provides a method of producingdonor vascularised tissue, suitable for transplantation into a recipientanimal in need of such treatment, comprising the steps of:

[0013] a) creating a functional circulation on a vascular pedicle in adonor subject;

[0014] b) partially or totally enclosing the vascular pedicle within afabricated chamber;

[0015] c) seeding the chamber with isolated cells or pieces of tissue;

[0016] d) implanting the chamber containing the vascular pedicle into ahost animal at any site where such an anatomical construct can becreated; and

[0017] e) leaving the chamber in the implantation site for a periodsufficient to allow the growth of vascularised new tissue.

[0018] In one preferred embodiment, the method comprises the step afterstep (a) of surrounding the vascular pedicle with added extracellularmatrix and/or a mechanical support. In another preferred embodiment, themethod comprises a step after step (b) of adding growth factors, drugs,antibodies, inhibitors or other chemicals to the chamber.

[0019] Preferably in step (e) the chamber is left in the implantationsite for at least 4 weeks, more preferably at least 6 weeks.

[0020] The vascularised tissue may be grown in vivo or in vitro, or maybe in situ in the host.

[0021] More preferably the chamber is implanted in the donor body,beneath the skin, although it is not limited to subcutaneous insertion.While externalization of the chamber during tissue/organ growth istheoretically possible, the high risk of infection makes this a rarelyused alternative.

[0022] For the purposes of this specification, the term “donor subject”is taken to mean an animal, especially a mammal and most especially ahuman, in which the donor vascularised tissue is created. For thepurposes of this specification, the term “recipient animal” is taken tomean an animal, especially a mammal and most especially a human, thatreceives the donor vascularised tissue graft. It would be appreciated bythose skilled in the art that as the generation of new vasculature,angiogenesis, in all warm blooded animals is associated with essentiallythe same physiological and pathological processes, methods disclosedherein are directly applicable to all warm blooded animals. The donorsubject is preferably a mammal, and may be a human or a non-humananimal. Preferred mammals include rodents, felines, canines, hoofedmammals such as horses, cows, sheep and goats, pigs, and primates. In aparticularly preferred embodiment, the donor subject and recipient arehuman.

[0023] The person skilled in the art will appreciate that a “vascularpedicle” is an artificial or naturally occurring arrangement of bloodvessels or vessel replacements that comprises an artery taking blood tothe site of the construct and a vein carrying it away. Preferably thevascular pedicle comprises an arterio-venous (AV) loop or shunt. In anAV loop or shunt the artery is either joined directly to the vein orconnected via a graft of a similar diameter so that there is noimpediment to blood flow (for example as illustrated in FIG. 1). In onealternative arrangement, the artery and vein are both ligated and bloodflow is via microscopic connections between the two (for example asillustrated in FIG. 3). In another alternative the artery and vein arein a “flow through” configuration with the blood vessels entering at oneend of a semi-closed chamber and exiting at the opposite side (forexample as illustrated in FIG. 4).

[0024] It would be appreciated by those skilled in the art that the term“functional circulation” as used herein describes a circulation that hasat least one of the following properties: the vessels making up thecirculation are patent, the vessels are capable of sustaining blood orblood-substitute flowing through them, the vessels are capable ofsupplying nutrients and/or oxygen to nearby tissue and the vessels arecapable of forming new blood vessels by budding.

[0025] Optionally, the chamber may also be supplied with addedextracellular matrix, for example matrix deposited by cells in situ,reconstituted basement membrane preparations such as Matrigel™ orlaminin (mouse origin), Amgel™, Humatrix™, or laminin (all of humanorigin) with or without matrix metalloproteinase inhibitors,polylactic-polyglycolic acid variants (PLGA), fibrin or plasma glue(autologous or heterologous) with or without fibrinolysis inhibitors, ornative collagen (autologous or heterologous) with or without collagenaseinhibitors.

[0026] In a preferred embodiment, extracellular matrix-likepolylactic-polyglycolic acid sponges, Dexon™ sponges, or sea sponges areadded to the chamber. Combinations of matrices, such as PLGA spongescoated with one or more other matrix-forming components such as fibrin,laminin, fibronectin, collagen, low molecular weight hyaluronan andvitronectin are other preferred options. Freeze dried segments oftissues such as muscle or organs such as liver may be used as sources ofmatrix and growth factors. Preferably the segments of tissues or organsare taken from the same species as the donor subject, and mostpreferably taken from the donor individual.

[0027] In a particularly preferred embodiment of the invention, thedonor subject is the same individual as the recipient animal, i.e. thegraft is autologous. Alternatively the donor subject may be animmunocompromised animal, such as an athymic mouse or pig, and therecipient may then be a different individual, i.e. the graft isheterologous. Other permutations and combinations of these proceduresmay include the use of either autologous or immunocompromised bloodvessels, cells, tissue segments or growth factors implanted back intoeither the original donor or a different recipient individual. Whetheror not the “maturity” of the graft confers immunoprotection on aheterologous graft is another variant that can be tested using routinetechniques.

[0028] The tissue or cells used in the chamber may be supplemented withadditional growth factors selected from the group consisting of “homing”factors to attract stem cells from the circulation, exogenous grabfactors such as α-Fibroblast Growth Factor (αFGF or αFGF-1),β-Fibroblast Growth Factor (βFGF-1 or βFGF-2), Platelet-Derived GrowthFactor (PDGF), Vascular Endothelial Growth Factor (VEGF-A,B,C,D or E),Angiopoietin-1 and -2, insulin-like Growth Factor (IGF-1), BoneMorphogenic Protein (BMP-2 and -7), Transforming Growth Factor-α and -β(TGF-α, TGF-β), Epidermal Growth Factor (EGF), Connective Tissue GrowthFactor (CTGF), Hepatocyte Growth Factor (EGF), Human Growth Hormone(UGH), Keratinocyte Growth Factor (KGF), Tumour Necrosis Factor-α(TNF-α), Leukemia Inhibitory Factor (LIF), Nerve Growth Factor (NGF),Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and otherfactors such as 3-isobutyl-1-methylxanthine (IBMX), insulin,indomethacin, dexamethasone, hyaluronan hexasaccharide, the PPAR-γligand Troglitazone, nitric oxide, prostaglandin E1, transferrin,selenium, parathyroid hormone (PTH), parathyroid hormone related peptide(PTrP), etc, many of which are promoters of angiogenesis orvasculogenesis. Antibodies, agonists or antagonists to some of thesegrowth factors or inhibitors of the chemical mediators can also be usedto influence the type of tissue formed and the rate of its formation.The person skilled in the art will readily be able to test which growthfactor(s), anti-growth factor antibodies, or inhibitors, or combinationthereof, are most suitable for any given situation.

[0029] The chamber may be used with autologous or heterologous cells,such as myoblasts transfected with Myo-D to promote formation of theskeletal muscle phenotype, stem cells with appropriate differentiationfactors, keratinocytes seeded to produce thin skin constructs for faceand neck reconstruction, etc. optionally the chamber may also compriseisografted or autologous cells selected from the group consisting ofmyoblasts, fibroblasts, pre-adipocytes and adipocytes, cardiomyocytes,keratinocytes, endothelial cells, smooth muscle cells, chondrocytes,pericytes, bone marrow-d rived stromal precursor cells, embryonic,mesenchymal or haematopoietic stem cells, Schwann cells and other cellsof the peripheral and central nervous system, olfactory cells,hepatocytes and other liver cells, mesangial and other kidney cells,pancreatic islet β-cells and ductal cells, thyroid cells and cells ofother ndocrine organs.

[0030] Alternatively the chamber may be used with additional autologousor isografted portions of skeletal or cardiac muscle, pancreas, liver,epididymal and other subcutaneous fat, nerves (peripheral, bloodvessel-associated, etc), kidney, bowel, ovary, uterus, testis, olfactorytissue or glandular tissue from endocrine organs. For the purposes ofthe specification the term “pieces of tissue” shall be taken toencompass any aggregates of cells, with or without additionalextracellular material such as extracellular matrix, either takendirectly from an animal or produced as a result of manipulation of cellsin tissue culture, or a combination of the two. In other variants suchtissue segments may be rendered ischaemic, cell-depleted or necrotic inorder to provide cues or signals to the surviving stem cells and othercells which may influence tissue development.

[0031] Depending on the nature of the supplementation provided to thecells, the vascularised tissue is enabled to differentiate in aparticularly preferred embodiment, stem cells, together with appropriateextracellular matrix and growth factor supplements, are supplied to thechamber in order to produce vascularised, differentiated tissues ororgans. Suitable pluripotent stem cells can be derived from:

[0032] a) blood;

[0033] b) bone marrow;

[0034] c) specific organs or tissues, including mesencymal stem cells;

[0035] d) cultured cells, which may be transfected or differentiated; or

[0036] e) placental stem cell banks.

[0037] To date we have used sourc s such as bone marrow, ischaemicskeletal muscle, and subcutaneous adipose tissue. Other potentialsources of pluripotent stem c lls are blood, especially from a fetus ornewborn individual but also from an adult, and human placenta. A numberof stem cell banks such as bone marrow or cord blood banks are alreadyestablished. Human embryos are a potential clinical source of stemcells, although legal and ethical issues precludes their use at presentin some countries.

[0038] The type of differentiated cells produced depends on the originof the stem cells, the local environment, the presence oftissue-specific growth or differentiation factors, and other factors.For example, unexpectedly we have observed that ischaemic skeletalmuscle placed in the chamber with an AV loop differentiates intopredominantly adipose tissue after 4-6 weeks. Without wishing to belimited by any proposed mechanism, we believe that in this case,mesenchymal stem cells in the muscle, together with the stimulus ofacidic ischaemic metabolites, are potentially responsible for thisdifferentiation. The chief advantage of using stem cells is their hugeproliferative capacity, so that relatively few cells are required togenerate a large colony for seeding the chamber and the AV loop.

[0039] Preferably the vascular pedicle, such as an AV loop comprises anartery joined to a venous graft, which is in turn joined to a vein.Alternatively the AV loop comprises an artery joined to a vein directly,or the AV loop comprises an artery joined sequentially to a venousgraft, an arterial graft, and a vein. In another variant, which isuseful where microsurgical anastomosis of vessels is technicallydifficult or impossible, a pedicle comprising the ligated stumps of anartery and vein (eg. the femoral vein) placed side by side in thechamber can be used as the blood vessel supply. In another preferredembodiment of the invention, the AV loop vessels flow in and out of thechamber from the same edge. In another variant the artery and vein areneither divided nor formed into a shunt, but instead flow in one side ofthe chamber and out the opposite side (see, for example, FIG. 4). In athird variant suitable for extremely small blood vessels, the artery andvein are divided and placed side by side in the chamber, the vesselsboth entering from the same edge; this is illustrated in FIG. 3.

[0040] The graft portion of the AV loop may be derived from the host orfrom a separate donor. Cold-stored or prefabricated vessels may also beused.

[0041] In one preferred embodiment of the invention, an additional stepinvolves the incorporation of a nerve stump, so that tissue in thechamber may become innervated. Skeletal muscle, for example, requiresproximity to a nerve for its maintenance and maturity; otherwise it willatrophy.

[0042] Preferably the chamber containing the vascular pedicle has adefined internal dimension. The internal dimensions, volume, and shapemay be varied in order to influence the volume and shape of the newtissue being produced. For example:

[0043] a) the internal volume of the chamber may be increased, withoutaltering the external size of the chamber, by providing thinner walls;

[0044] b) the shape of the chamber may be constructed to resemble thatof the target organ or body part, such as an ear, nose, breast,pancreas, liver, kidney, finger or other joint;

[0045] c) the degree of permeability of the walls of the chamber may bevaried; for example the chamber may include a semi-permeable membranecomponent to allow selective perfusion of molecules into and out of thechamber, or a plurality of perforations may be placed in the walls ofthe chamber to allow an increased flow of metabolites and metabolicby-products, growth factors and other factors that influence cellsurvival, growth and differentiation between the inside and outside ofthe chamber. The size, shape and number of the perforations may beselected according to the size of the donor vascularised tissue and therequirement to keep the contents of the chamber isolated from directcontact with the implantation site. Alternatively,

[0046] d) a semi-permeable component may be placed within the chamber inorder to isolate “feeder” cells from immune reactions.

[0047] As an example of the latter, populations of fibroblasts r othercells can be transfected, then used as a source of the transfected geneproduct(s) within the chamber. This construct is placed within asemi-permeable pocket out of contact with the host's immune system. Drugdelivery is used to switch the transfected gene on or off. These cellswill survive by diffusion as long as they receive adequate nutrients,but will eventually die.

[0048] The surface chemistry of the chamber walls may be modified, inorder to modify the interaction between the tissue and the chamber wall,to provide a stimulus for differentiation or to incorporate or be coatedwith a gel, such as alginate, which mediates the slow release of achemical or biological agent to create a gradient.

[0049] The degree of internal support within the chamber may be varied,eg there may be:

[0050] a) no support;

[0051] b) a solid support which directs, encourages or inhibits thegrowth of the new tissue, or excludes new tissue, or is incorporatedinto the new tissue;

[0052] c) a transient support based on resorbable materials;

[0053] d) a porous supporting material which supports cell and vascularingrowth, providing a skeleton over which the new tissue can begenerated, eg sponge-like materials such as blown PTFE materials, PLGAsponges of variable composition and porosity, etc;

[0054] e) a support formed from materials which direct tissuedifferentiation, such as hydroxyapatite or demineralised, granulatedbone.

[0055] Preferably the exterior surface of the chamber bears a means bywhich the chamber can be attached and/or immobilised to the desiredregion of the body.

[0056] In a second aspect, the invention provides a vascularised tissugraft, ie. the contents of the chamber, comprising differentiated tissueor an organ with a mature vascular supply.

[0057] Preferably the graft predominantly comprises tissue selected fromthe group consisting of adipose tissue, cartilage, bon , skeletalmuscle, cardiac muscle, loose connective tissue, ligament, tendon,kidney, liver, neural tissue, bowel, endocrine and glandular tissue.More preferably the graft predominantly comprises vascularised adiposetissue, skeletal muscle, cartilage or bone tissue or tissue comprisingpancreatic islet and/or ductal cells, kidney cells or liver cells.

[0058] In a third aspect, the invention provides a method of repairing atissue deficit, comprising the step of implanting a tissue chamberaccording to the invention into a patient in need of such treatment, inwhich:

[0059] a) the tissue or “organ” graft is formed according to the methodsof the invention, and;

[0060] b) retained for sufficient time to mature ie. to achieve thedesired size, vascularity and degree of differentiation, and;

[0061] c) transferred to the desired recipient site; and

[0062] d) the blood vessels of the graft are microsurgically anastomosedto a local artery and vein.

[0063] For the purposes of the specification, the term “tissue deficit”will be taken to comprise a shortfall in the normal volume, structure orfunction of a tissue in the recipient. Such a tissue may be selectedfrom, but is not limited to superficial tissues such as skin and/orunderlying fat, muscle, cartilage, bone or other structural orsupporting elements of the body, or all or part of an organ. Theaugmentation of otherwise normal tissues for cosmetic purposes, such asforms of breast augmentation, is also provided by the invention. Aperson skilled in the art will readily recognise that such a tissuedeficit may be a result of trauma, surgical or other therapeuticintervention, or may be congenitally acquired.

[0064] In a fourth aspect, the invention provides a method of providinga subject with a gene product, comprising the steps of:

[0065] a) constructing a tissue chamber according to the invention tocreate vascularised tissue from a patient in need of such therapy;

[0066] b) removing the chamb r with its vascularised tissue andculturing the chamber assembly in vitro;

[0067] c) transforming cells of the tissue in the chamber with a desiredgene; and

[0068] d) implanting the chamber or the contents minus its chamber intothe patient.

[0069] The timing of the genetic transformation of the tissue-producingcells can be varied to suit the circumstances, for example the cells maybe transformed at the time of setting up the chamber construct, duringthe incubation, or immediately prior to transplantation.

[0070] The provision of gene products can take several forms. Oneexample is the transfection of myoblasts with the Myo-D gene to createtissue with a normal skeletal muscle phenotype. Such transfected cellsmay then be seeded into the desired chamber, matrix and AV loop togenerate vascularised skeletal muscle. This may have implications forthe treatment of muscular dystrophy and other genetically inheritedmuscle diseases. A second example is the transfection of pancreaticislet cells with a “healthy” phenotype and their seeding into thechamber. This approach may prove to be useful in the treatment ofdiabetic patients. In a third example, cells are transfected with agrowth factor gene or an angiogenesis-promoting gene, such as PDGF, bFGFor VEGF, prior to seeding them into the chamber together with the AVloop and selected matrix. This continuous production of growth factor isdesigned to speed up the rate of development of, and the rate of newblood vessel formation within, the new tissue/organ.

[0071] In a fifth aspect, the invention provides a model system forvascularised tissue, comprising a tissue chamber containing a vascularpedicle of the invention and optionally an extracellular matrix,operably connected to an extracorporeal circulation apparatus and renaldialysis filter. The extracorporeal circulation apparatus and renaldialysis filter may be of any suitable conventional type. The cellsforming the tissue in the chamber are optionally transformed so as toexpress a heterologous gene. This model system may be used forculturing, recruiting, growing and studying the behaviour of stem cellsor tissue containing precursor cells, either in vitro or in vivo.Because of the ability to alter the environment of the chamber withadded growth, differentiation and chemical factors, it is possible toproduce a wide variety of tissues and organs by this process.

[0072] The ability to generate autologous vascularised tissue of adefined composition and at any anatomical site in the body where it ispossible to create an arterio-venous loop or suitable vascular pediclehas many other applications. At its localised site the tissue in thechamber may, for example, be manipulated by

[0073] a) gene transfection,

[0074] b) administration a local drug or other “factor”, or

[0075] c) creating a site of circulatory stem cell homing.

[0076] Furthermore, the tissue and exudate in the chamber may readily beharvested to monitor progress of tissue growth and development. Aboveall, it is the ability to grow and transplant new vascularised,differentiated tissues or organoids that sets this invention apart fromothers.

[0077] For the purposes of this specification it will be clearlyunderstood that the word “comprising” means “including but not limitedto”, and that the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

[0078]FIG. 1 illustrates how the femoral artery and vein are anastomosedmicrosurgically to a vein graft of similar diameter to form a loop(shunt). The AS loop is placed as shown in a plastic chamber (made ofpolycarbonate or poly-L-lactic acid, etc), the lid secured, and thechamber optionally filled with an extracellular matrix with or withoutadded cells or growth factors. The chamber is anchored in positionrelative to the surrounding tissue by means of stay sutures throughexternal holes.

[0079]FIG. 2 shows a configuration similar to FIG. 1, except that thelid of the chamber is dome-shaped and the edges of the chamber are morerounded to minimise wound breakdown.

[0080]FIG. 3 depicts an example of the thin-walled chamber used for thepedicle model. In this case an artery and a vein are ligated distallyand placed adjacent to each other. Microscopic connections between theartery and vein become established, and form an AV loop in a similarmanner to that shown in FIGS. 1 and 2.

[0081]FIG. 4 shows a model chamber similar to that in FIG. 3, but withexit holes for the blood vessels at either end of the chamber. Thisallows an undivided, dissected length of blood vessels, placed side byside, and in some variants surrounded with extracellular matrix, to formnew tissue.

[0082]FIG. 5 shows the inner aspect of an AV loop-containing chamber, 7days after insertion. Fluorescence microscopy shows labelled fibroblastsevenly distributed across the chamber surface, magnification×160 (seeExample 2).

[0083]FIG. 6 shows a reconstructed “breast” on a male rabbit,constructed using a vascularised, tissue-engineered fat and connectivetissue flap created at a remote site (the groin region) in the samerabbit (see Example 10).

DETAILED DESCRIPTION OF THE INVENTION

[0084] The invention will now be described in detail by way of referenceonly to the following non-limiting examples and drawings.

[0085] Experimental Procedures

[0086] Preparation of Tissue Chamber

[0087] A custom-made polycarbonate chamber was pr pared. It has a topand a bottom, and when the two halves are sealed together th internalvolume is 0.45-0.50 ml. The general construction of the chamber isillustrated in FIG. 1.

[0088] The basic chamber for use in rats is made of polycarbonate. Inone variant the chamber is made of polylactic acid or PLGA. The chamberis in the shape of a cylinder of external dimensions 14 mm diameter and4 mm high, with a saw cut on one side to create an opening for the bloodvessel entry and exit. Another variant has cut openings on oppositesides of the chamber to allow blood vessels to flow in one side and outthe other. The chamber has a base and a removable lid. The base hasholes to allow anchoring of the chamber to subcutaneous tissue. Theinternal volume is approximately 0.45-0.50 ml. The internal volume ofthis basic chamber can be varied, maintaining the same external volume,by using thinner walls, which may even be as thin as a standard plasticfilm used in food storage. An alternative design is in the shape of a“dome” with more rounded edges, as shown in FIG. 2. Other variantsinclude an elongated, flattened cigar shape as shown in FIG. 3 whichfits readily into the subcutaneous space in the groin. For the purposesof specific grafts, the shape of the chamber may be designed to mimicthe shape or contours of a particular body part, for example a humanfinger joint or thumb, human ear, human nose, human breast, etc.

[0089] The size of the chamber can be scaled up or down to suit the sizeof the host. Hence the internal volume for a chamber to be used in amouse may be approximately 0.1-0.2 ml, in a rabbit 10-12 ml, but in ahuman can be up to approximately 100-200 ml.

[0090] The chamber may optionally be sealed. In the standard version theopening allows limited contact with the surrounding tissue and totaluninterrupted contact with the blood supply. In a sealed variant, theopening is engineered to allow just enough space for the ingoing arteryand outflowing vein without crushing the blood vessels. The vessel portsare sealed, for example with fibrin glue, to avoid contact of thedeveloping graft with sounding tissue.

[0091] The surface of the polycarbonate chamber can be left in itsnative hydrophobic state, or can be rendered relatively more hydrophilicby the use of polylactic acid or the pre-treatment of polycarbonate witha thin film of poly-L-lysine. In one useful configuration, the surfaceof the chamber comprises a plurality of perforations, allowing increasedcontact with growth factors in the surrounding tissue. The size andshape of the perforations may be tailored to optimise the passage of thedesired factors, while minimizing or preventing the passage of cells.

[0092] If the chambers are made of glass or Pyrex they can be coatedwith silicone.

[0093] The chamber design should ideally fit comfortably into therecipient site, and should be of a rounded shape and of a sufficientlysmall size to avoid wound break down.

[0094] The internal contents of the chamber are sufficiently large toaccommodate an osmotic pump (eg. an Alzet™ osmotic mini pump) to deliverdrugs, growth factors, antibodies, inhibitors or other chemicals at acontrolled rate. In one alternative method of drug/factor delivery, theosmotic pump may be placed subcutaneously outside the chamber with aplastic tube leading from the pump placed inside the chamber, eg. at thecentre of the AV loop.

[0095] Creation of an Arteriovenous (AV) Shunt Loop Inside the TissueChamber

[0096] The basic model has been described by Tanaka et al (1996).Briefly, male Sprague-Dawley rats (225-285 g) were anaesthetised withintraperitoneal phenobarbitone (50 mg/kg; 2.5 ml of a 6 mg/ml solution).Under sterile conditions an inferior-based flap was created in the rightgroin to expose the femoral vessels from the inguinal ligament to thesuperficial epigastric branch. A longitudinal incision was made in theleft groin to harvest the left femoral vein from inguinal ligament tothe superficial epigastric branch. This vein graft (approximately 1.5-3cm long; usually 2 cm) was interposed between the recipient rightfemoral vein and artery at the level of the superficial epigastricartery by microsurgical techniques using 10-0 sutures. The shunt wasplaced into the chamber, the lid closed and the construct sutured to thegroin musculature with the aid of small holes on th base of the chamber.An adipose layer was placed over the chamb r and the wound closed with4-0 silk sutures.

[0097] The growth chambers with the AV shunts were harvested at either2, 4 or 12 weeks post implantation.

[0098] Assessment of Vascularisation and Tissue Creation

[0099] At the specified time of exploration, the chamber was opened, andthe vessels cleaned and tested for patency. The vessels were tied offwith a 5-0 silk suture at the entrance of the chamber and the flapharvested. In 2 of the 5 rats in each group the flap was perfused, viathe aorta, with India ink prior to harvest (details below). The flapswere assessed for volume and weight and placed in buffered 10% formalsaline (BFS) for histological examination. The animals were sacrificedwith an intracardiac dose of sodium pentabarbitone (˜3 ml of 250 mg/mlsolution) at the completion of the exploration.

[0100] Tissue Mass and Volume

[0101] The tissue in the chamber was removed and its wet weight andvolume recorded. The volume of the tissue was assessed by a standardwater displacement technique. The tissue was suspended by a 5-0 silksuture in a container of normal saline which had been zeroed previouslyon a digital balance. Care was taken not to touch the container with thespecimen. The weight recorded was the volume of the tissue specimen(with a density equal to that of normal saline, 1.00 g/ml). The mass ofthe specimen was assessed at the same time on the same digital scale byallowing the tissue to rest on the base of the container, and recordingthe weight.

[0102] India Ink Perfusion

[0103] In order to perfuse the flaps with India ink, the abdomen wasopened via a midline incision. The intestines were gently retracted tothe periphery and the periaortic fat stripped away. The proximal aortaand inferior vena cava were ligated. The aorta was cannulated with a22-gauge angiocatheter which was secured with a distal suture around theangiocatheter and aorta. A venotomy was carried out in the inferior venacava. The aorta was perfused with 10 ml of heparinised saline to flushout the retained blood, the animal was sacrificed with intracardiacsodium pentabarbitone (3 ml of a 250 mg/ml solution), the aorta infusedwith 3 ml buffered 10% formol saline (BFS) and then with 5 ml India inkin 10% gelatin. The flap vessels were then tied off. Tissue from thechamber was removed, fixed in BFS, cleared in cedar wood oil and thepattern of vessels visualised microscopically using transmitted lightand image analysis (Video Pro™ imaging).

[0104] Histology

[0105] Specimens were fixed in buffered formol saline and embedded inparaffin. Sections (5 μm) were cut and stained with either haematoxylin& eosin (H & E) or Masson's Trichrome.

EXAMPLE 1 Creation of Vascularised Tissue in Chambers with an AV Loop

[0106] Three groups of five rats each were used. Each group had anidentical procedure performed as described above, and the growthchambers with the AV shunts were harvested at either 2, 4 or 12 weekspost implantation.

[0107] The average mass of the AV shunt vessels prior to insertion was0.020 g (exsanguinated) and 0.039 g (when full of blood). Two weeksafter insertion the AV shunt and its surrounding tissue weighed0.18±0.03 g. The mass increased progressively being 0.24±0.04 g at 4weeks and 0.28±0.04 g at 12 weeks. The volume of the new tissue closelyparalleled its weight. The increase in weight but not volume between 2and 12 weeks was statistically significant (P<0.05, ANOVA/Dunnett'stest).

[0108] Two weeks after implantation the AV loop was surrounded by a massof coagulated exudate containing varying amounts of clotted blood. At 4weeks the mass of tissue around the loop was larger and firmer,especially in its central part. By 12 weeks the newly formed tissuesurrounding the loop had increased still further in volume and nowfilled approximately two-thirds of the chamber. The surface coagulum wasno longer visible, and the whole mass had a uniformly firm consistency.

[0109] After 2 weeks of incubation the AV shunt was surrounded by a cuffof newly-formed connective tissue composed of fibroblasts, thin collagenfibres and vascular sprouts, arranged roughly vertical to the shunt.Inflammatory cells, both neutrophils and macrophages, were present inmoderate numbers in the outer part of the newly formed tissue and in thesurrounding mass of coagulated inflammatory exudate. In occasionalsections, branches of newly-formed blood vessels arising from the venouslumen of the AV shunt could be identified.

[0110] In the 4 weeks incubation group, the newly formed tissue was moremature. The zone closest to the AVS contained a dense plexus of newlyformed vessels embedded in mature collagenous stroma. Outside this layerwas a less mature zone similar to the newly formed tissue in the 2 weeksspecimens. Most of the surrounding coagulum was no longer visible, andonly small numbers of inflammatory cells were present in the newlyformed tissue. As at 2 weeks, communications between the AV shunt andthe newly formed vessels were visible in some sections.

[0111] Twelve weeks after incubation, the newly formed tissue hadmatured still further, and consisted of dense collagenous connectivetissue with fibroblasts aligned parallel to the outer margin of the AVshunt. There was no apparent decrease in vascularity and newly formedvessels formed a dense plexus throughout the connective tissue. Fewinflammatory cells were visible.

[0112] At all three time points, the specimens which were injected withIndia ink gave a clearer picture of the extent and density of the newlyformed vasculature. In most specimens almost all vessels containedcarbon in their lumen, indicating that they communicated with the AVshunt.

[0113] Ideally, newly formed tissue must be stable and capable ofretaining its shape. The tissue formed around an AV loop has both thesecharacteristics. At 2 weeks the mass within the chamber is soft andreadily deformed. By 4 weeks it is firmer and more rigid, and at 12weeks it has the physical characteristics of mature connective tissue.Surprisingly, growth is continuous for at least 12 weeks afterimplantation, with no indication of resorption or regression of thenewly formed tissue with increasing maturity.

EXAMPLE 2 Chambers with Rat Dermal Fibroblasts

[0114] Culture of Rat Dermal Fibroblasts

[0115] Rat skin was harvested in a 6 cm by 4 cm ellipse from the groinarea of an inbred Sprague-Dawley rat line (Monash University AnimalServices, Clayton, Victoria, Australia). The inbred line comprisedanimals resulting from at least 20 generations of brother-sistermatings.

[0116] The epidermis was trimmed off. Segments of dermis were cut into 2m=by 2 mm squares and 10 pieces were placed onto a sterile Petri dishand attached to the base using rat plasma “glue”. This glue was made bythe addition of 2 ml of rat plasma, prepared from Sprague Dawley rats,to 0.3 ml of 2% calcium chloride. The glue was allowed to set for 10 minat 37° C. Complete culture medium, comprising Dulbecco's Modification ofEagle's Medium (DMEM), 10% fetal calf serum, penicillin and streptomycinand glutamine, was added to the culture dish. The skin segments wereleft undisturbed for 7 days, then the medium was changed. There wasconsiderable outgrowth of fibroblasts by 10 days, at which time the skinsegments were removed. The fibroblasts were subcultured twice at weeklyintervals, each time growing the cells in 75 cm² and 175 cm² cultureflasks respectively.

[0117] The fibroblasts were labelled with two fluorescent labels,bisbenzamide (EB) and carboxyfluorescein diacetate (CFDA). Three ml of0.1% trypsin in phosphate buffered saline (PBS) at pH 7.4 was added to a175 cm² cell culture flask containing confluent fibroblasts for 5 min at37° C. The trypsin was neutralized by the addition of 17 ml of completeDMEM media. The cell suspension was centrifuged at 2000×g for 10 min.The cell pellet was resuspended in 3 ml of media and the suspensiontransferred in three 1 ml aliquots to Eppendorf tubes. To each Eppendorftube 13.5 μl of a 10% CFDA solution and 20 μl of BS were added. Thetubes were incubated for 1 h at 37° C. and shaken gently every 15minutes. The cells then were transferred into a 175 cm² flask andrecultured. CFDA persists in the cytoplasm of cultured cells andsurvives the division of cells into daughter cells. CFDA fluorescesmaxmimally at 513 nm; BB fluoresces maximally at >430 nm. Labelled cellswere protected from light, in an effort to maintain maximalfluorescence.

[0118] Cell Counting

[0119] Prior to the addition of cells to the chambers, the fibroblastculture flasks were trypsinized and the trypsin neutralized. 10 μl ofsuspended cells were counted using a hemocytometer, and 0.05% Evan'sblue dye in a 1:10 ratio. The solution was centrifuged and the resultingcell pellet suspended in an appropriate volume of bovine collagensolution to yield a cell concentration of 1 million cells/ml.

[0120] Rat Tail Tendon Collagen (RTTC)

[0121] The tendons from six rat tails were harvested and diced into2×2×2 mm cubes (yield approximately 10 g). Four hundred ml of cold 0.5 Macetic acid was added and the mixture homogenized and left stirring at4° C. for 24 h. The homgenate was centrifuged (3000 rpm×20 min) and thesupernatant harvested. This extraction procedure was repeated twice withfurther additions of 300 ml of cold 0.5 M acetic acid. To the pooledextracts a solution of 5 N NaCl was added slowly, with magnetic stirringat 4° C., until the final concentration of salt was approximately 0.7 M(100 ml of 5M NaCl added to every 600 ml of extract). The solution wasleft for 1 h to allow full precipitation of the native collagen. Theprecipitate was collected by centrifugation (3000 rpm×20 min at 4° C.),redissolved in 200 ml of 0.5 M acetic acid and dialysed twice against 2l of cold 0.5 N acetic acid for 24 h, and twice against sterile, colddistilled water, the final dialysis solution containing a few drops ofchloroform on the surface. This results in a sterile stock solution ofRTTC of approximately 3 mg/ml, the concentration checked by a Bradfordprotein assay (Bio Rad) with a Type I collagen standard.

[0122] Preparation of Chambers

[0123] All procedures were carried out in a cell culture hood using strile technique. Chambers were coated internally with RTTC by addition of200 μl of 2.5 mg/ml RTTC solution, pH 7.4, to each half chamber.Chambers were incubated for 1 h at 37° C. to allow gel formation anddried for 24 h. After rinsing with PBS to remove residual salt crystals,0.25×10⁶ of fluor scently labelled fibroblasts in 150 μl of complete MMwere added to each half chamber. After allowing 1 h for adherence of thecells, chambers were immersed in complete DMEM and incubated at 37° C.under 5% CO₂ in air for 24 h. The density of labelled of cells wasdetermined by counting the number of cells in 7 randomly selected fieldsof each half chamber using a ×10 objective.

[0124] Insertion of Chambers

[0125] Two groups of 6 inbred male Sprague-Dawley rats, weighing between230-280 g, were used. Two chambers were inserted into the inguinalregion of each rat, the chamber in the right side containing an AV shunt(prepared as described above) and that in the left side containing noshunt. In 6 rats chambers were removed 2 days after implantation. Theremaining 6 chambers were removed 7 days after implantation.

[0126] Examination of Chambers After Removal

[0127] The chamber was removed, the AV shunt examined for patency andthe flap removed. Ten μl of 0.05% Evan's blue dye was added to each halfchamber and incubated for 5 min at 37° C. The base of each half of thechamber was then examined, using a ×10 ocular, to determine the numberof Evan's blue-stained and fluorescent cells in 7 randomly selectedmicroscopic fields. The number of labelled cells in 7 random fields onthe surface of the AV shunt was then determined.

[0128] Two days after insertion the shunt and surrounding tissue coveredapproximately 20% of the surface of the chamber; by 7 days this hadincreased to approximately 30%. On this basis the overall density ofcells in the chamber containing an AV shunt was calculated by summationof the density of cells on the surface of the chamber and 20% (2 days)or 30% (7 days) of the labelled cells on the surface of the AV shunt.

[0129] Paired t-tests were used to compare number of cells per grid inthe control and experimental chambers and the preop rative number ofcells per grid using Microsoft Excel™ and Graph Pad Prisms software (SanDiego, Calif., USA).

[0130] After counting, the shunt and surrounding tissue was fixed in 10%formol saline, embedded in methacrylate and thin sections prepared andstained with either haematoxylin and eosin or Masson's trichrome.

[0131] Comparison Between Labelling with Bisbenzamide (BB) andCarboxyfluorescein Diacetate (CFDA)

[0132] In both in vitro cultures and the in vivo chambers the number anddistribution of labelled cells at the two wavelengths examined (430 nmfor BB; 573 nm for CFDA) was the same. No cells were identified as beinglabelled with only one fluorescent dye. Hence in the results whichfollow “fluorescent cells” refers to cells labelled with both BB andCFDA.

[0133] Macroscopic Findings

[0134] The AV loop was patent in every chamber.

[0135] Two days after insertion the AV shunt covered approximately 20%of the surface of the chamber. By 7 days the area covered by the AVshunt and new tissue arising from it had increased to approximately 30%.

[0136] The 2 day mean weight of the shunt was 0.12±0.017 g and the meanvolume was 0.12±0.014 ml. By 7 days the mean weight had risen to0.23±0.018 g and the mean volume to 0.21±0.015 ml.

[0137] Density of the Labelled Cells

[0138] The density of the labelled cells in empty and AV shuntcontaining chambers is shown in Table 1. TABLE 1 Density of labelledcells. (mean number/grid) in empty and AV shunt containing chambers,pre-operatively and 2 and 7 days after insertion. AV Shunt-containingTime after Pre- Empty chamber Insertion operative Chamber In chamberTotal* 2 days  8.6 ± 1.74  4.0 ± 0.94  4.8 ± 0.59  5.7 ± 0.62 7 days10.2 ± 1.7 4.8 ± 1.3 11.7 ± 1.4 15.5 ± 1.1·

[0139] It can be seen that in all chambers the cell density decreased inthe early stages after implantation, the values in all 2 day chambersbeing less than their pre-insertion density. Two days after insertionthere was no significant difference in the density of cells in empty andAV shunt-containing chambers.

[0140] At 7 days the density of the cells in empty chambers did notdiffer significantly from the density 2 days after insertion. Incontrast, the cell density in AV shunt containing chambers increased toalmost 3 times its 2 day value, and both the density of cells in thegrid and the density (after allowing for the number of labelled cells inthe tissue surrounding the shunt) were significantly greater than thedensity in empty chambers (p=0.013).

[0141] Evan's blue staining showed that in all chambers examinedvirtually all labelled fibroblasts were viable, with less than 1% ofcells taking up the Evan's blue dye.

[0142] Histological Findings

[0143] After 2 days incubation the vessels of the AV shunt weresurrounded by blood clot and coagulated inflammatory exudate. Smallnumbers of fibroblasts were visible migrating from the vascularadventitia into coagulum.

[0144] By 7 days, many more fibroblasts were present within thecoagulum, and early vascular sprouts were visible arising from the outeraspect of the AV shunt.

[0145] At both 2 and 7 days fluorescent studies showed labelledfibroblasts on the surface of the coagulum surrounding the AV shunt, butlabelled cells were not seen within its substance. The inner aspect ofan AV shunt-containing chamber removed 7 days after insertion is shownin FIG. 5.

EXAMPLE 3 Differentiation of Stem Cells in Implanted Tissue Chambers

[0146] Skeletal muscle, pancreas, fat, liver and kidney were asepticallyremoved from four inbred Sprague-Dawley rats. They were chopped into 1mm cubes and placed in a tissue culture-grade petri-dish (15-20 pieceseach 7 cm² of culture surface) containing 1-2 ml of complete serum-freeDMEM. They were then incubated for a minimum of 24 h and up to 3 days.At the appropriate time 4-6 pieces of tissue were adhered in a plasmaclot to each side of a chamber of the type described in Example 2. Thechamber was then seeded with the AV loop and closed. The proximal end ofa femoral nerve was placed inside one half of the chambers containingskeletal muscle explants. After 4-6 weeks the rats were sacrificed andthe chambers examined.

[0147] After 4-6 weeks, the contents of chambers with tissue explantsdiffered from the contents of chambers without tissue explants, in thatthey contained new and different cell phenotypes. In all cases most ofthe necrotic tissue explants had been replaced by clumps of new cells.

[0148] In the most dramatic of these experiments, 8 of the 11 chambersseeded with skeletal muscle explants contained up to two thirds of theirvolume with mature, well-vascularised adipose tissue together withmature skeletal muscle fibres, surrounded by a thin capsule. The matureregion of the new tissue contained up to 90% vascularised adiposetissue. The remaining chambers also had a lesser proportion of matureadipose tissue and skeletal muscle fibres.

[0149] The chambers seeded with portions of pancreatic tissue had alarge population of well-demarcated large ovoid eosinophilic cells, manygiant cells and other smaller cells.

[0150] Without wishing to be limited by any proposed mechanism, webelieve that a “stem cell” population, either attracted into the chamberfrom a circulating stem cell source by the necrotic tissue explants, orcontained within the tissue explants, has given rise to the new tissue.In either case a very small amount of explant tissue was used, incomparison to the large amount required to isolate stem cells, and ourresults indicate that this is a novel and efficient method to obtainstem cells. The stem cells may have differed with respect to theirdegree of commitment to a particular tissue type, or else they may haveresponded to cues expressed by the unique microenvironment of thedifferent explants, to proliferate and differentiate into the differentcell types observed.

[0151] The generation of encapsulated adipose tissue described here is,to our knowledge the first time that such a neo-organoid has been grownde novo on its own artery and vein.

[0152] A detailed study of the spatio-temporal and dynamic changes inthe chamber and the mechanism by which these events give rise to theneo-organ may also have applications in defining in vivo stem cellavailability and behavior. The chamber model is superior to any other invivo model available so far, since it enables a wide variety ofmanipulations of the chamber contents and environment and stem cellsources. Furthermore, it enables a study of stem cells in a naiveenvironment without the influences of other nearby tissues, as opposedto the growth of stem cells in an established tissue.

[0153] The finding that muscle explants can result in the generation ofa neo-organ, consisting almost entirely of mature adipose tissue,indicates that:

[0154] a) a stem cell population can successfully seed the chamber;

[0155] b) the chamber model supports the plasticity of stem cells;

[0156] c) a satisfactory, appropriate and adequate neovascularisationdevelops with, integrates and supports the tissue construct;

[0157] d) the constructs are not overcome by fibroblastic in-growth; and

[0158] e) the constructs are not overcome by inflammatory cells.

[0159] These results demonstrate that application of the chamber modelto tissue engineering is feasible, and represent a significant advancein the art of “tissue engineering”.

EXAMPLE 4 Effect of Matrigel

[0160] A pilot study was devised to determine if there was any initialloss of Matrigel during 20 minutes of contact with the AV loop. Based onthe results of the pilot study, time periods of 2, 4 and 8 weeks werechosen. At the 4 week time period a further comparison was done withgrowth factor-reduced Matrigel. Six male Sprague-Dawley rats were usedper group, each weighing between 220 and 280 g. The arterio-venous loopprocedure was carried out as described in the Experimental Procedures.

[0161] Matrigel (Collaborative Research Inc, Bedford, Mass., USA) wasdivided into in sterile 10 ml aliquots at an approximate concentrationof 12 mg/ml in DMEM containing 10 μg/ml of Gentamycin (BectonDickinson). The Matrigel was stored at −20° C. and prior to use wasthawed overnight at 4° C. Throughout the preparation process theMatrigel was kept on ice and manipulated using pre-cooled pipettes.Growth factor reduced (GFR) Matrigel was prepared from matrigelessentially as described by Vikicevic et al (1992). This involved anadditional fractional ammonium sulphate step. The protein concentrationof the resultant GFR Matrigel was verified by Bradford protein assay andby Coomassie blue staining after SDS-PAGE to be consistent with that ofnormal growth factor-replete Matrigel.

[0162] Under sterile conditions, 0.5 ml of Matrigel was added to eachsterile chamber at room temperature where it gelled rapidly (within 15seconds). The chamber with matrigel was then placed in position in therat's right groin. The Matrigel is gelatinous at room temperature,enabling immersion of the loop within it. In the pilot study the AV loopwas made and immersed in the Matrigel for 20 minutes beforeimplantation, to d termine whether there was any initial loss ofMatrigel from the chamber due to liquefaction of the matrix.

[0163] For the time course studies the new tissue flaps were harvestedat 2, 4 and 8 week periods. The flaps were harvested at the above timeperiods, and assessed for weight, volume and histology. Statisticalanalysis was carried out comparing the 2, 4 and 8 week groups with eachother and the AV loop alone (See Example 1). A further comparison wasdone at 4 weeks between Matrigel, GFR Matrigel and the AV loop alone at4 weeks.

[0164] In the pilot study Matrigel proved easy to manipulate in vitro.There was minimal loss of Matrigel after 20 minutes of contact with theAV loop.

[0165] In an AV loop alone (no added matrix), the average weight of thenew tissue flap formed after 4 weeks was 0.24±0.04 g, and the averagevolume was 0.23±0.03 ml. These results acted as the control for thisexperiment and Example 5.

[0166] At two weeks the average weight of flap in chamber supplementedwith Matrigel was 0.32±0.03 g and volume was 0.30±0.03 ml. This wassignificantly greater then the 4 week loop alone flap (p=0.05). At fourweeks the flaps were slightly heavier than the 2 week flaps, with anaverage weight of 0.35±0.03 g and a volume of 0.33±0.03 ml. A comparisonof these two groups showed no statistical significance. The weight(p=0.01) and volume (p=0.01) were both significantly greater than thecontrol flaps produced by loop alone.

[0167] At 8 weeks the flaps had regressed, with an average weight of0.18 g±0.02 g and volume of 0.16 ml±0.02 ml. Statistical analysisreveals that this is highly significant in weight (p=0.002) and volume(p=0.001) when compared with both the two week flaps and the four weekflaps weight (p=0.0005) and volume (p=0.0003). For this longer timecourse 8 rats were operated on to compensate for infection ordehiscence. No such problems were encountered, so all 8 have beenincluded in the analysis.

[0168] The GFR Matrigel flaps were smaller than the normal Matrigelflaps at 4 weeks, weighing on average 0.27±0.02 g. A comparison ofweights showed no statistical significance. The volume was 0.24±0.01 ml;this was significantly less than the normal Matrigel (p=0.04). The GFRflaps were still larger than the loop alone at the same time period (notstatistically significant). One of the chambers became infected, and hadto be removed. As a consequence there were 5 animals examined in thisgroup.

[0169] At 2 and 4 weeks a significant flap of tissue had formed whencompared to chambers containing the loop alone at day 0. There wasresidual Matrigel in the chamber, and strands of microvessels werevisible running from the flap edge into the Matrigel. Microfil injectiondemonstrated good filling of flap vessels, including the advancingmicrovessels. This appearance was not apparent at 8 weeks, when theflaps were smaller and with a more regular smooth surface. At 8 weeksthere was only residual fluid in the chamber, and no viscous Matrigelwas visible.

[0170] Histological examination showed that at 2 weeks there were manyimmature vessels extending to the flap edge, with haemorrhage within theperipheral tissue. There was early collagen formation in the centralportion and areas of unincorporated Matrigel within the flap.

[0171] At 4 weeks the vessels had matured into arterioles and venules,with larger branching vessels arising from the loop and smaller branchesat the periphery. There was still some unincorporated Matrigel and smallamounts of haemorrhage. The unincorporated Matrigel contained sparsefibroblasts and the occasional vessel. The general impression was of amaturing but still growing flap with good vessel formation.

[0172] At 8 weeks the flap tissue appeared more mature, with densercollagen and larger vessels nearer the loop. It was less cellular withless vessels. A capsule had started to form around the generated tissue,and there was residual Matrigel remaining within the flap.

[0173] The GFR Matrigel flaps appeared to be more mature, with largervessels in the centre and less active angiogenesis at the periphery.There was evidence of early capsule formation and in some specimens moreinflammatory cells were present.

[0174] At all time courses Microfil injection demonstrated good vascularconnection between the loop and the flap vessels.

EXAMPLE 5 Effect of Poly-L-lactic Polyglycolic Acid (PLGA)

[0175] (a) Ply Prepared by the Salt-Leached Method.

[0176] A PLGA insert for the tissue chamber was constructed using aparticulate leaching method as described by Patrick et al (1999). Inessence PLGA is dissolved in chloroform and mixed with NaCl. Afterevaporation of the chloroform the resulting scaffold is machined to thedesired shape. The salt was then leached from it leaving interconnectedpores. The pore size is a reflection of the size of the salt particleused. In this experiment pores of 300-400 μm and a porosity of 84% weremade. The PLGA was machined in two parts so as to fit inside thepolycarbonate chamber. The lower part comprised a base plate containinga groove for the loop and the upper part comprised a flat disc to coverthe loop and base plate. The PLGA discs were 1.4 mm in diameter by 2.5mm thick. The PLGA was sterilised and pre-wetted by soaking in 100%alcohol for 30 minutes on a mechanical stirrer then subjecting them tothree 30 minute washes in sterile saline washes, also on a mechanicalstirrer.

[0177] The arteriovenous loop was prepared as described above, andplaced into the base plate of PLGA sitting in the chamber. The superiordisc was placed on top and the chamber closed. Each group of ratscontained 6 male Sprague-Dawley rats, with each rat weighing between 220and 280 grams. The chambers were harvested at either 2 or 4 weeks.Weight, volume and histology were assessed at both time periods.Immunohistochemical staining of flap sections for α-actin was carriedout to detect myofibroblasts. In each group, one chamber was excluded,one due to infection and the other to dehiscence, leaving 5 rats in eachgroup.

[0178] At 2 weeks the vessels had almost entirely vascularised theconstruct, with some uninvolved PLGA at the tip. The capsule had begunto form proximally near the portal. At 4 weeks the construct wasentirely encapsulated, and had shrunk and retracted, withdrawing fromthe sides of the chamber. Micro-fill injection demonstrated the extentof vessel penetration.

[0179] The 2 week flap weight was 0.43±0.05 g and the volume 0.38±0.04ml. The 4 week flap weight was 0.33 g±0.04 g and the volume 0.29 ml±0.04ml. A comparison between the 2 and 4 week groups showed a reduction inflap size between 2 and 4 weeks. This result was not statisticallysignificant. Further comparison with other experiments was not possibledue to the presence of PLGA retained within the flap, which skewed theresults.

[0180] At both 2 and 4 weeks there was extensive vessel outgrowth, withbranching vessels found up to the edge of the PLGA. Arterioles hadformed, and healthy branching angiogenesis was seen coming from theloop. The cellular infiltrate was lying on the matrix and on the surfaceof the structure. A capsule had formed on the proximal part of the flaponly at 2 weeks. α-Actin stain showed that this capsule containedmyofibroblasts. At 4 weeks the capsule was thicker proximally, with moremyofibroblasts and had extended to encompass the whole flap.

[0181] (b) PLGA Prepared by a Fiber-Spun Method.

[0182] The vascular loop model described in Example 1 was used in thisexperiment. The AV loop was placed within a round polycarbonate chamber(0.5 ml volume) filled with a PLGA disc (75% poly-L-lactic acid/25%polyglycolic acid) as the scaffold. The PLGA scaffold was eithermanufactured by the salt leaching method described above or a fiber spuntechnique. Each group comprised five animals. After 4 weeks incubationand immediately before harvest heparinised India Ink was infused i.v.for 5 min. Tissue from the chamber was harvested, fixed in buffered 10%formalin, paraffin embedded, cut into 5 μm sections and stained withhaematoxylin & eosin (H & E) for evaluation.

[0183] The salt-leached PLGA was less dense than the hard, denseconsistency of the fiber-spun PLGA. This was evidenced by the subsequentcutting of the tissue/PLGA blocks for histological evaluation. Thesalt-leached PLGA was brittle and prone to crumbling. The fiber-spunPLGA was easy to section as it had a solid consistency and did notcrumble.

[0184] Histological examination showed a consistent pattern for allspecimens in their respective groups. In the salt-leached PLGA group,considerable invasion into the PLGA by microvasculature and new tissuewas found throughout, with numerous India Ink filled microvesselsevident. The fiber-spun PLGA differed in character. Theneovascularization and new tissue formation developed predominantly in atwo dimensional plane. Initially, instead of invading the PLGA, tissuepreferentially surrounded the PLGA discs and migrated towards the edgeof the chamber. Tissue invaded the matrix at a much slower rate. Oncethe edge of disc was reached further thickening of new tissue grewaround the disc but not completely engorging it after 4 weeks.

[0185] Further modifications to the fibre-spun PLGA, such as increasingthe pore size and decreasing the density (and therefore the hardness)may make this technique a viable alternative to the salt-leached PLGApreparation.

EXAMPLE 6 Model System for Vascularized Tissue

[0186] The tissue chamber and graft system of the invention may be usedas a model to examine the behaviour of vascularised tissue, through theuse of an extracorporeal circulation machine to maintain the developingtissue in vitro during its generation. The chamber contents areestablished as specified in Example 1. The host's blood or suitabletransfused blood (at least 90 ml) is taken and heparinised (up to 50units/ml). The blood vessel ends are connected to silicone tubing andthe blood is oxygenated via a renal dialysis filter. The oxygenatedblood is pumped through the tissue using conventional intensive careunit instrumentation adapted for this purpose, and maintained in vitroin this manner until the tissue/organ is mature. During this phase bloodsamples are constantly monitored to assess the degree of coagulation andthe maintenance of haemostasis. In a similar manner to the in vivostudies, genetic modification of the tissue generating cells can beapplied to this model. Finally the tissue/organ generated ismicrosurgically replaced into the appropriate site in the host. A majoradvantage of this method is the ability to produce tailor-made,off-the-shelf parts and organs.

[0187] The next step in testing our model is to add stem cells to thesystem and see whether tissue is generated de novo. The isolation,expansion and seeding of “stem cells” into the chamber is a huge areafor research in itself and is still in its infancy. For various reasons,we have chosen an unorthodox method of adding stem cells andenvironmental cues, with unexpected results. We have investigated thebehaviour of injured/necrotic tissue explants placed in vivo in thechamber, and have demonstrated conversion of muscle into fat (seeExample 3).

[0188] The hypothesis being tested in experiments such as these is thatthese small tissue explants may harbour at least a few stem cells, whichperceive an injury to their parent organs and respond by initiatingtissue renewal. We have also tested a number of tissues, including fat,liver and kidney, and will shortly investigate neural, uterus, ovarian,thyroid and glandular tissue. The results have been very promising,because all of the tissues tested have “driven”, by unknown mechanisms,the generation of a cell phenotype not normally present in the chamber.Mechanistically they have converted the cellular/angiogenic response inthe chamber from one analogous to “inflammation and scar formation”,involving the de novo generation of tissue largely composed offibroblasts, to one analogous to “tissue renewal and generation”, alsoknown as “scarless” tissue repair in the fetus, comprising thegeneration of vascularised tissue with a recognisable three dimensionalorganisation and phenotype. Significantly, the new tissue formed is freeof fibroblastic in-growth and of inflammatory cells.

EXAMPLE 7 Assessment of Hypoxia Within the Tissue Growth Chamber

[0189] For the study of hypoxia of the cells within the chambers, AVshunt loops were created in anaesthetised male rats as previouslydescribed in Example 1. Standard-sized chambers (0.5 ml volume) wereused. Chambers were filled with Matrigel, as described in Example 5, andseeded with immortal rat L6 myoblasts (1×10⁶ cells/0.5 ml Matrigel)distributed over the entire surface area. Chambers were then positionedin the groin of the rat.

[0190] Chambers were harvested at 3 days, 7 days, and 2 and 4 weeksincubation. At the time of exploration the animals were again ansthetised with sodium phenobariton (30 mg/ml) and an assessment ofanoxia was made by injection of nitroimidazole (60 mg/kg, i.p.) 2 hoursbefore the time of chamber harvest: Rats were sacrificed with a lethaldose of pentobarbitone sodium (3 ml of a 325 mg/ml solution) afterharvesting the chambers. Specimens within the chambers were processedfor histology and immunostaining with nitroimidazole antibody. Underthese circumstances, the only cells which label are those which arehypoxic (<10 mm Hg) and which are proliferating.

[0191] An assessment of the degree of Oxygenation of tissue at days 3and 7 showed proliferating, hypoxic cells in the immediate vicinity ofthe vascular loop at both time points. After 2 weeks the only labelledcells were at the periphery of the growing mass of new tissue. By 4weeks, no cells were labelled with nitroimidazole.

[0192] The results from this study indicate that a state of hypoxia andactive biosynthesis exists in cells close to the blood vessel loop. Thisstrongly suggests that hypoxia is a driving force of angiogenesis in thepolycarbonate chamber particularly in the first week. Those cells remotefrom the AV loop were undoubtedly hypoxic but were not proliferating.During week 2 the hypoxic, proliferating cells were located in theadvancing edge of the new tissue, but by the end of week 4 the chamberwas well oxygenated throughout and new tissue formation had slowedconsiderably. Studies such as this enable the researcher to invetigatehow hypoxia can influence the growth of new tissue within the chamber.

EXAMPLE 8 Isolated, Cultured Cells Added to Chambers in the Rat AV LoopModel

[0193] (a) Addition of Myoblasts to Chambers

[0194] Skeletal muscles from various parts of the body (eg.gastroenemius, rectus femoris, latissimus dorsi, etc) were harvestedfrom neonatal rats 5 days after they were weaned. Myoblasts weregenerated from this harvested tissue by collagenase digestion andculturing in Ham's F10 culture medium containing 20% fetal calf serumwith 2 ng/ml of bFGF. Myoblasts were identified by desminimmunostaining. Fibroblasts were removed by serial subculturing, takingadvantage of the fact that they adhere to plastic within half an hourwhereas myoblasts adhere after that time. Enriched myoblasts (2-4×10⁶cells) were inserted into either (1) Matrigel alone (approximately 0.5ml) or (2) Matrigel (approximately 0.15 ml) with PLGA making up thebalance of the volume. These matrices were placed around an AV loopwithin a standard 0.5 ml chamber, as previously described. Theseconstructs were incubated subcutaneously for either 2, 4, 6, 12 or 16weeks. At the time of exploration, the rats were placed under generalanaesthesia, and the tissue formed within the chamber (also known as the“flap”) was removed. Approximately half of the tissue was frozen inisopentane and the other half fixed in formalin, and sectioned, prior tomorphological, histological and immunohistochemical staining.

[0195] 1. Matrigel Only Group

[0196] Group A—2 Weeks (n=6)

[0197] The chambers from six rats were examined at 2 weeks. There was alarge amount of muscle in four of these; and of these, 3 containedidentifiable desmin-positive myoblasts and evidence of myotubeformation. The other two contained no desmin-positive tissue.

[0198] Group B—6 Weeks (n=9)

[0199] Of the 9 rats in this group, 2 constructs contained muscle andmyotubes, 4 flaps contained no identifiable muscle, and 3 rats diedprematurely.

[0200] Group C—12 Weeks (n=11)

[0201] Of the 11 rats in this group, no constructs contained muscle, 5flaps contained no muscle but did contain some (as yet identified)tissue, 2 chambers contained no flap (possibly because it slipped out ofthe chamber) and 3 rats died prematurely.

[0202] 2. PLGA/Matrigel Group

[0203] Group A—2 Weeks (n=3)

[0204] No results for this group.

[0205] Group B—6 Weeks (n=6)

[0206] Of the 6 rats in this group, 2 constructs contained muscle andmyotubes, and 4 flaps contained no muscle. In one chamber in which themyoblasts were fluorescently labelled with CFDA prior to being seededinto the chamber, there was evidence of myoblasts still surviving after4 weeks' incubation in vivo.

[0207] Group C—12 Weeks (n=7)

[0208] Of the 7 rats in this group, 2 constructs contained ds-in-stained myoblasts, 5 flaps contained unidentified tissue but nomuscle, and 1 rat died prematurely.

[0209] Group D—16 Weeks (n=5)

[0210] No results for this group.

[0211] In H&E stained sections of flaps after 2 weeks incubation,myoblasts were evident in some tissue specimens, with their presenceconfirmed by immunostaining for desmin. Within 2 weeks, groups ofmyoblast nuclei had aligned and formed into myotubes which stainedpositively for dystrophin and formed mature striated skeletal muscle. By6 weeks, myotubes and mature muscle were present in some specimens butconnective tissue formed in others. At both 2, 4 and 6 weeks mononuclearleukocyte infiltrate was present, probably due to the use of Matrigel,which originates from mouse cells. However, by 12 weeks, much of theflap tissue was resorbed. Interestingly, in some of the earlyexperiments with “less pure” myoblasts seeded, isolated pockets ofosteoid (bone tissue) and adipose tissue (fat) were also observed after2 and 4 weeks in the Matrigel only experiments.

[0212] In preliminary experiments, a femoral nerve severed distally wasincorporated into Matrigel matrix, adjacent to the loop and surroundedby the seeded myoblasts (n=6, 2 weeks incubation). There appeared to bea trend towards reduced desmin-positive muscle cells (compared with thenerve-free controls, Group 1A) but there was positive immunostaining forS100, a Schwann cell marker, in most of the newly generated tissue.

[0213] We know from previous work that this model provides a goodangiogenic stimulus, and we have mow shown that this model can sustainthe survival, expansion and differentiation of myoblasts. Thevascularised chamber can also support this cell line and provide anoptimal environment in which the chosen cell can differentiate in anormal and expected fashion. Histological evidence demonstrates that theseeded myoblasts both survive and differentiate to form myotubes, whichin turn coalesce to form mature skeletal muscle in this model, over aperiod as short as 2 weeks.

[0214] (b) Stem Cell Addition

[0215] Using the same AV loop model, we have investigat d the fate ofgreen fluorescent protein (GFP) labelled and non-labelled rat bonemarrow-derived stem cells into these chambers.

[0216] Bone marrow-derived stromal cells were harvested from rat femursby flushing them with normal saline. These cells were then labelled andsorted on a FACS machine. The stromal cell subpopulation was expanded byculturing in a α-MEM medium containing 20% fetal calf serum. Theexpanded cells were retrovirally transfected with Green FluorescentProtein (GFP) and a neomycin plasmid to enable them to be tracked withinour flap. When sufficient cells were available we placed them at aconcentration of 2×10⁶ per 0.5 ml Matrigel into our AV loop chambermodel.

[0217] Nine AV loops in chambers containing these stem cells wereconstructed using either Matrigel alone (n=8) or Matrigel/PLGA (n=1) andthe matrix. Rats have been examined at 2 weeks (n=4) or 4 weeks (n=4).In frozen sections some fluorescence is seen in these specimens,although it is not clear whether this is genuine GFP fluorescence orautofluoresence. In subsequent experiments the resultant tissue from ourGFP-labelled flaps has been cultured in the presence of neomycin-richmedia. Surviving GFP-labelled cells have been detected under suchconditions after 2 and 4 weeks in the chamber, whereas non-GFP-labelledcells failed to survive under these conditions. However, to date we havefound no evidence of specific tissue phenotype or clone formation in newtissue arising from these seeded cells.

EXAMPLE 9 Pancreatic Cells Added to Chambers in the Rat AV Loop Model toForm a Transplantable Pancreatic Organoid

[0218] All experiments were performed using inbred Sprague-Dawley rats.The experimental model used of an arteriovenous (AV) fistula createdwith a vein graft in the right groin and placed within a 0.5 ml internalvolume polycarbonate chamber, was consistent throughout all experimentalgroups.

[0219] Rats were anaesthetised with pentobarbitone prior to surgery asdescribed in previous examples. Pancreatic tissue for transplantationwas prepared by various methods:

[0220] (a) “Ficoll islets”: Using adult donor rats, the isolatedpancreas was digested with collagenase P (Boehringer Mannheim, Germany)in vitro, and the islets purified by centrifugation on a Ficoll densitygradient.

[0221] (b) “Histopaque islets”: Using adult donor rats, the vasculatureof the pancreas was perfused in vivo with 7 ml of collagenase(Worthington Biochemicals, USA) at 1.3 U/ml. The resultant islets wereisolated and purified using Histopaque [Liu and Shapiro, 1995].

[0222] (c) “Digested pancreas”: Using adult donor rats, the isolatedpancreas was digested with collagenase P (Boehringer Mannheim, Germany)in vitro, but the preparation was not subjected to any furtherpurification step.

[0223] (d) “Filtered pancreas”: Using adult donor rats, the isolatedpancreases were not enzymically digested but simply homogenised and thecrude extract sieved through a range of different sized filters. Thefraction which passed through the 450 μm filter but was retained by the100 μm filter was used in further experiments.

[0224] The extracellular matrix used as a support for seeding the isletpreparations were used in one of the following configurations:

[0225] (i) The chamber was filled with Matrigel, and the islets weredispersed throughout.

[0226] (ii) The chamber was filled with Matrigel and theislets/pancreatic tissue was placed in centre of chamber/AV loop.

[0227] (iii) 150 μl of Matrigel containing the islets/pancreatic tissuewas placed in centre of chamber in close proximity to the AV loop.

[0228] (iv) 150 μl of rat plasma clot containing the islets/pancreatictissue was placed in centre of chamber in close proximity to the AVloop.

[0229] The experimental groups were devised as follows:

[0230] Group 1. Old (400-500 g) inbred Sprague Dawley rats were used.“Ficoll islets” were placed in Matrigel. There were 3 recipient rats. Weused a 2.5:1 (donor:recipient) ratio, and 10-17 days incubation.

[0231] Group 2. Old (400-500 g) inbred rats were used. “Digestedpancreas” were placed in Matrigel. There were 3 recipient rats. We useda 1:1 (donor:recipient) ratio, and 11 days incubation.

[0232] Group 3. Adult (230-260 g) inbred rats were used. “Digestedpancreas” was placed in Matrigel. There were 6 recipient rats. We used a1:1 (donor:recipient) ratio, and 7-14 days incubation.

[0233] Group 4. Adult (230-260 g) inbred rats were used. “Histopaqueislets” were placed in Matrigel. There were a recipient rats. We usedboth 1:1 and 4:1 (donor:recipient) ratios, and 6-21 days incubation.

[0234] Group 5. Adult (230-260 g) inbred rats were used. “Filteredpancreas” was placed in a plasma clot. There were 8 recipient rats. Weused a 1:2 (donor:recipient) ratio, and 8-24 days incubation.

[0235] In Vitro Experiments

[0236] Islets were kept in culture in Matrigel, with DM media changestwice weekly, in parallel with the above in vivo experiments to test thelongevity of islets in culture. Insulin immunostaining was performed onseveral such cultures at one and two months with positive stainingresults.

[0237] Serum Insulin Level Measurements

[0238] At the time of chamber harvest, blood samples (100 μl) were takenfrom the loop artery and vein and systemic venous circulation, formeasurement of insulin levels by radioimmunoassay for the rat isoform.

[0239] Chamber Harvest and Flap Manipulation

[0240] Chambers were harvested at the above time points, and tissueswere preserved in Buffered Formal Saline and routine histologicalpreparation, followed by paraffin embedding. Histological s ctions weresubject d to routine (H&E) and immunostaining (for insulin andglucagon).

[0241] In Vitro Culture

[0242] Survival of islets was demonstrated to 4 and 8 weeks in culture.H&E and insulin staining showed functional survival at these timepoints. The islet clusters had begun to dissociate into individual cellsand clumps of cells between 4 and 8 weeks.

[0243] Serum Insulin Levels

[0244] Serum insulin levels in were tested in experimental groups 3 and4 described above. Venous (outflow) blood exhibited serum insulin levelsthat were 30-50% lower than those in the arterial (inflow) serum in mostanimals. In two animals, levels were 40% and 100% higher in the venoussystem.

[0245] Analysis of the Chambers

[0246] Tissue in the chambers was divided into four parts and serialsections made. Large amounts of angiogenesis and collagen depositionwere confirmed, in keeping with the original model. H&E stainingdemonstrated occasional islet persistence in all groups, but not in allflaps. Inflammatory infiltrates were present in most flaps, consistingmainly of lymphocytes. Ductal elements were observed in the Group 5“filtered pancreas” chambers, although no confirmatoryimmunohistochemistry was performed. Insulin and glucagonimmunohistochemistry demonstrated occasional positive staining,particularly for glucagon.

[0247] These experiments demonstrate that the AV loop chamber modelcreates a suitable environment to support the survival of islets in asignificant number of the constructs for periods up to 24 days. Insulinand glucagon production was identified by immunostaining in histologicalsections of tissue during this same period. However, the long termviability of this new “organoid” and its continued insulin productionremains to be evaluated.

EXAMPLE 10 Increasing the Amount of Tissue in the Rat Model Through theuse of Larger Chambers.

[0248] (a) Rat Experiment

[0249] The amount of tissu produced in the rat using the standardchamber model (˜0.3 ml) is quite substantial in comparison with theanimal's body size, and corresponds to a small “breast” or small “organ”within the body. In order to be able to reproduce this finding in thehumans it is essential to test the limits of tissue production. This canbe don firstly in the rat, through the use of larger volume chambers.Therefore, the aim of this study was to assess whether larger amounts oftissue could be grown over a longer period of time (4-8 weeks) insidelarger chambers. In this fashion it is proposed that this method can beused to produce clinically useful amounts of new tissue which, ifnecessary, could be transferred on its own vascular pedicle to anotherpart of the same individual.

[0250] The basic model of the arteriovenous (AV) shunt loop in anenclosed growth chamber has been described in detail in Example 1. TheAV shunt was placed within a dome-shaped chamber (FIG. 2). The chamberwas made of polycarbonate, had a proximal opening for the pedicle andconsisted of abase plate and a lid. It had a base diameter of 17 mm, acentre-of-base to top-of-dome distance of 1.3 mm and an internal volumeof 1.9 ml. In contrast, the standard chamber described in previousstudies (for instance ales 1 and 2) had a volume of 0.5 ml. The AV shuntwas sandwiched between two custom-made disks of PLGA which was used as amatrix to fill the chamber. The PLGA was prepared according to the saltleaching method described by Patrick et al.(1999). Pore sizes between300-420 nm and a porosity of 80-90% was achieved. The disks weresterilised by four cycles of mechanical stirring for 30 minutes in 100%ethanol, then three times sterile, phosphate buffered saline, beforeuse.

[0251] After positioning the lop in the chamber the lid of the chamberwas closed and the chamber embedded beneath the inguinal skin andsecured with three 6-0 prolene holding sutures. The wound was closedwith 4-0 silk sutures. Chambers were harvested from rats under generalanaesthesia at 2, 4, 6, and 8 weeks incubation for further analysis (n=6per group). Th animal was finally killed by an overdose of Lethobarb (3ml) administered by intracardiac injection.

[0252] Whole mount specimens were fixed in buffered formal saline (BFS)and cut into 1 mm slices. Half of these slices, in alternating order,were embedded in paraffin and stained with H&E for histologicalcomparison of the maturity of the newly formed tissue and itsvasculature. The other half of these slices were stored in 100% ethanoland used for point counting on a grid to assess the percentage of thenewly formed tissue, the remaining PLGA, and the AV loop in thespecimen. Every fifth field of 100 points was counted on the front andback of each tissue slice. For this purpose, the slices were dipped inhaematoxylin briefly before counting. This enabled newly formed tissueto be readily distinguished from PLGA. The results of point counting onthe grid enabled calculation of the percentages of newly formed tissue,remaining PLGA, and AV loop and comparisons of those values at 2, 4, 6,and 8 weeks. Statistical differences between newly formed tissue weightand PLGA weight in time were calculated using Student's t-test withp<0.05 being statistical significant.

[0253] Weight and volume measurements: All specimens harvested from thechambers were assessed for volume and weight. The volumes of thespecimens, as measured by fluid displacement, was not statisticallysignificant different from the measured weights. The total averageweight (equivalent to volume) of the specimens decreased progressivelyin time. The total average weight % standard deviation (SD) of eachgroup of specimens was 1.07±0.06, 1.03±0.06, 0.96±0.06, and 0.81±0.18grams, at 2, 4, 6 and 8 weeks, respectively. This resulted in astatistically significant decrease of specimen weight between timepoints apart 4 weeks or longer, which may be accounted for by theprogressive gradual resorption of PLGA matrix.

[0254] The amount of PLGA and tissue in the specimen was studied toassess their involvement in the overall decrease in weight of thespecimens. All specimen were point counted microscopically with the aidof a grid to determine the percentage of specimen taken up by PLGA ortissue. The decrease in specimen wet weight was attributed to resorptionof PLGA. The total average weight of PLGA±SD at 2, 4, 6, and 8 weeks,respectively, was 0.89±0.07, 0.56±0.14, 0.34±0.07, and 0.20±0.09 g. Onthe other hand the newly formed tissue component of the specimen showeda progressive increase of weight in time. The total average weight oftissue±SD at 2, 4, 6, and 8 weeks, respectively, was 0.13±0.04,0.42±0.09, 0.57±0.06, and 0.58±0.10 g. The increase in tissue weight wasstatistically significant over all consecutive time periods, except forthe period between 6 and 8 weeks (P<0.05). Over this 6-8 week period,tissue growth reached a plateau, although it also did not decrease asnoticed in previous experiments in smaller sized chambers filled withPLGA (Example 5.).

[0255] Macroscopic findings: After India-ink injection,neovascularisation could be readily identified during processing of thetissue. New vasculature did not reach the outer edge of the PLGAscaffold at any time point. However, in one serendipitous finding, achamber was inadvertently left incubating in a rat for 10 months. Whenharvested, the chamber was totally full of soft connective tissue, whichwas well vascularised and had patent blood vessels supplying nutritionto the tissue “flap”.

[0256] (b) Rabbit Pilot Experiment

[0257] Preliminary results from the rat experiments indicated that thelarger chambers were able to grow more tissue and for a longer periodthan the standard chambers. Where the walls of the large chambers wereperforated with numerous holes, a further improvement in the rate of newtissue growth, the amount of tissue produced and growth to the edges ofthe chamber were found [Tanaka Y, 2000, unpublished findings]. Theselatter conditions approach the optimal conditions for tissue growth inthis model. The major aim of this pilot study was to assess whethertissue production could be scaled up in an animal which is 8-10 timesthe size of a rat, and whether the tissue would maintain its size andshape.

[0258] The experimental model used was the basic AV shunt loop in anenclosed growth chamber, however the experimental animal was the NewZealand White rabbit.

[0259] Pre-operative analgesia was given in the form of carprofen (1.5mg/kg, s.c.). New Zealand White rabbits (2.0 to 2.8 kg) wereanaesthetised with i.v. pentobarbitone (30 mg/kg) and maintained in aface mask with halothane and oxygen (2.0 L/min). Under sterileconditions a graft of 4-6 cm (rabbits) respectively was harvested fromthe left femoral vein, and used to create an AV shunt between theproximal ends of the divided right femoral artery and vein. The AV shuntwas placed within a dome-shaped chamber, in this case made ofpolyurethane, with the approximate dimensions 3.0 cm diameter, 2.0 cmhigh, with an opening for the vessel entry and egress (FIG. 2). In someinstances the anatomy of the rabbit permitted the use of an AV pediclerather than an AV loop, because the small connecting vessels in thesurrounding tissue of the pedicle made it a naturally occurringflow-through loop. In this latter example the effect of the AV bloodflow was comparable but the operating time and postoperative pain wasless. In the usual configuration this chamber had a plurality of smallperforations in the chamber walls. Subcutaneous fat in the groin regionwas used as a source of adipocytes and adipogenic precursor cells.(Zuket al, 2001). The fat tissue was formed into a crude slurry by injectionthrough an 18 gauge needle. These cells were donated by and implantedinto the same rabbit.

[0260] The AV shunt loop or pedicle was placed within the chamber, whichwas filled with a 3-dimensional matrix made of a combination of PLGAwhich was machined to fit the chamber, Matrigel, Type 1 porcine skincollagen or a similar suitable composition, and the preadipocyte-richfat tissue slurry. The Matrigel was then allowed to gel. The lid wasclosed and the chamber embedded beneath the inguinal skin. The woundclosed with 4-0 nylon sutures.

[0261] Approximately 6-8 weeks later, with the animal again undergeneral anaesthesia, the chamber with its associated blood vessels wasremoved from the groin and the chamber. Two flaps have been analysed todate.

[0262] The tissue in the chamber was removed and its wet weightrecorded. The tissue was also be suspended by a fine cotton suturethread and wholly immersed in a beaker of water on a balance. The mass,assuming a density of 1.00 g/ml, is the tissue volume. Specimens werefixed in buffered formol saline (BFS), embedded in paraffin and stainedwith either ME or Masson's Trichrome (a connective tissu stain).

[0263] The volume of new tissue generated after 8 weeks growth was 10-11ml (compared with a total volume of the chamber estimated to be 12 ml).The composition of the flap was adjudged to be a mixture of adipose andother connective tissue. The shape was preserved when transferred underthe nipple of the same male rabbit and the volume sufficient to enablethe construction of a medium-sized breast on this animal (see FIG. 6).

[0264] We have achieved the production of clinically useful amounts oftissue in the rat and rabbit. The tissue thus produced was of a size andshape potentially suitable for breast reconstruction and similarapplications. Flaps such as these with their associated patent bloodvessels have the potential to be transferred to another part of the bodyfor reconstructive purposes.

EXAMPLE 11 A New Model of Vascularised Tissue Engineering in the Mouse

[0265] In order to investigate the fundamental processes of tissueengineering it is desirable to develop a suitable tissue engineeringmodel in the mouse for the following reasons:

[0266] Genetic Technology: Transgenic and gene knock-out technology ismuch further advanced in mice, allowing us to probe the influence of anumber of factors involved in tissue engineering such as growthpromoters and inhibitors.

[0267] Stem Cell Biology: Stem cells are pluripotent cells that giverise-to all tissues; they are highly durable and can thereforetheoretically resist the initially hostile ischaemic environment of thechamber. This makes them attractive cells to seed in the chamber. Stemcell biologists have cloned a wide variety of stem-cell sub-types inmice that can be seeded into the mouse model in order to attempt togenerate specific tissue types.

[0268] Cost: There are also significant cost benefits in using mice.Purchase, housing and caring for mice is less expensive than for largeranimals. Also there will be a reduction in the use of expensivelaboratory consumables such as growth factors.

[0269] We investigated two different types of vascular configurationsthat have been shown to be angiogenic in previous work, in order todetermine the best technique to use in the mouse. The first was a tiedoff arteriovenous pedicle (AVP) of the femoral artery and V in (Khouriet al, 1993; FIG. 3) and the second was a “flow through loop” pedicle(FTLP) configuration (Morrison et al, 1990; FIG. 4).

[0270] The polycarbonate chamber, when used in the rat model, did notadversely affect the patency rate of the high-flow microsurgicalarteriovenous loop. It was also tolerated well by these animals. Howeverthis material is hard and has sharp edges which was felt might affectthe patency rate in the mouse due to the lower flow rate of the proposedvascular configurations and smaller diameter vessels in this animal.Therefore polycarbonate chambers were compared with softer siliconechambers in order to determine the most suitable material to use in theconstruct of the chamber. We also compared the two main extracellularmatrices used in the rat model (Matrigel and PLGA) in the mouse to judgewhich was best with regard to angiogenesis and tissue growth. A total of88 C57BL/6 wild-type mice (male and female; 18-24 g body weight) wereused for this set of experiments.

[0271] Initially two vascular configurations were examined usingspecially constructed polycarbonate chambers. The first was a tied offAVP of the mouse femoral artery and vein as described by Khouri et al[1993] in the rat (FIG. 3). The second was a FTL pedicle comprising thesuperficial epigastric vessels encapsulated within a modified version ofthe polycarbonate chamber as described by Morrison et al [1990] (FIG.4). There were 3 groups of 6 animals for both vascular configurations.Each configuration was examined at the 2, 4 and 6 weeks. The experimentwas to be performed using both Matrigel® and PLGA as extracellularmatrices (n=2×2×3×6=72).

[0272] All operations were performed under general anaesthesia (chloralhydrate, 4 mg/g body weight, i.p.). The right groin and upper leg wererendered hair free using a combination of clipping and a depilatorycream. The skin was decontaminated using an alcohol preparation. Thetied off pedicle technique required a vertical incision extending formthe groin crease to the knee just offset from the saphenous vesselswhich are visible through the skin. The saphenous vessels were tied offat the kn e and then dissected free from their accompanying nerve backto the origin of the femoral artery at the inguinal ligament. Theflow-through model was performed using a transverse groin incision sitedjust above the groin fat pad. The superficial epigastric (SE) vesselswere dissected free of the surrounding tissue from their origin at thefemoral vessels for a distance of approximately 1 cm to their entry intothe groin fat pad. Here the vessels course through the fat pad sendingnutritional branches to the fat and glandular tissue around them. Theythen anastomose directly with an ilio-inguinal vessel (a direct branchof the infra-renal aorta) that pierces the abdominal wall at the lateralaspect of the inguinal ligament to enter the fat pad from the lateralside. The entire fat pad is mobilised free of the skin and underlyingmuscle thus creating a space into which the chamber will alter beintroduced. Thus the SE vessels have an arterial input and venousdrainage from both sides which we felt would augment the long termpatency rate in this model. To our knowledge this is the first time thatthis vascular arrangement has been described in the mouse. The first cmof the SE vessels (where they are free of the fat pad) is thenencapsulated in a modified polycarbonate chamber that is split down oneside and the appropriate extracellular matrix (Matrigel or PLGA) isinserted into the chamber. The chamber is then sealed at the proximalend and along the lateral split using melted bone wax (Ethicon bonewax™) taking care not to apply the heated wax directly to the vessels.The seal is augmented by two 10/0 nylon microsutures placed at eitherend of the lateral split and the whole chamber is anchored to theunderlying muscle near the origin of the SE vessels in order to preventthe pedicle from being dislodged during post-operative mobilisation. Asmall amount of fatty tissue surrounding the vessels as they enter thefat pad is allowed to “plug” the distal end of the chamber. This plug isthem augmented with wax sealant and the whole construct is carefullyplac d in the groin so that it lies in the dissected space lateral tothe femoral vessels. The wounds were closed using a combination ofburied interrupted horizontal mattress sutures and a running suture(both 6/0 silk) as these animals tend to gnaw at their wounds.

[0273] Following early analysis of the results in each group the tiedoff AVP group of the experiment was discontinued. This was because thethrombosis rate was unacceptably high (11/14 animals) and pursuit ofthis line of investigation seemed futile and wasteful of animals. Thisobservation contrasts with Khouri's work in the rat and our ownexperience in the rat and the rabbit where the tied-off AVP remainspatent in the majority of cases. The most likely reason for the highthrombosis rate in our study is that the mouse vessels are extremelysmall (internal diameter approximately 0.2 mm) and very sensitive todissection. Flow rates in vessels of this size are also very low. Thethrombosis rate in the FTLP group was better (3/11) but still seemedexcessively high.

[0274] We postulated that the polycarbonate material we were using wastoo hard and sharp for the delicate vessels of the mouse. Ourexperimental plan was modified to include a cohort of animals withchambers made from medical grade silicone (Animal Ethics committeeapproval was obtained for this modification). Two cohorts (1 with PLGAand 1 with Matrigel®) of 3 groups of 4 animals were used for thismodified aspect of the experiment (n=2×3×4=24) using only the flowthrough vascular configuration. Accurate estimation of the volume andweight of the specimens proved impossible. The volume of the chamber isapproximately 80-100 μl. This varies for several reasons such as theamount of wax or fat that encroaches on the entry points of the chamber.Also it is difficult to measure the exact volume of extracellular matrixthat is used in each chamber. Matrigel is usually added as a liquid andallowed to gel in vivo. Some spillage may occur during infusion orduring manipulation of the chamber. We also noted that the volume of theMatrigel declined by at least 50% over the first two weeks such that thespecimen that was removed was actually smaller than that inserted.

[0275] The weight of the PLGA used in each chamber could be accuratelymeasured but the volume was impossible to ascertain as the structure wasporous and had to be broken up into crumbs in order to easily fit itinto the small chamber. Given these inaccuracies we did not attempt toevaluate quantitatively the chamber tissue and looked at the morequalitative aspects of the device such as morphology of the newly formedtissue. Patency of the vessels was determined at microsurgicalexploration and via India ink perfusion studies. If the vessel wasextensively thrombosed within the chamber it was usually possible to seethis under the operating microscope. However ascertaining definitivepatency was not always possible. Therefore India ink perfusion studieswere performed under general anaesthesia on each animal prior tosacrifice. Under the operating microscope the groin incision wasreopened and the chamber exposed taking care not to damage the pedicle.A laparotomy was then performed and the abdominal aorta was dissectedfree of the vena cava and cannulated just below the renal vessels usinga fine (30G) bore silicone tube. This was then flushed using heparinisedsaline to ensure that the cannula was in the correct position. Next asolution of neat commercial India ink containing 10 i.u. heparin per mlwas infused under gentle hand pressure using in a pulsatile fashionuntil the animals liver had turned completely black. Previousdescriptions also advise the use of gelatin in this solution but in ourpreliminary trials of the technique we found that the gelatin formedclumps that obstructed the fine bore tube and resulted failure of theprocedure (this occurred even if the gelatin was warmed to bodytemperature prior to infusion). Patency could be confirmed under directvisualization of the transparent chamber as the India ink could clearlybe seen tracking into the chamber along the vascular pedicle. Followingthis the animals were sacrificed via a lethal overdose of phenobarbitoneand the chambers were carefully removed cutting the pedicle(s) flushwith their entry into the device.

[0276] The specimens were fixed in formalin and taken through gradedalcohol solutions to absolute alcohol. They were then immersed in methylsalicylate and allowed to clear over 72 hours. This allows directvisualization of the vascular tree which has been perfused with Indiaink. All specimens were then examined as whole-mount preparations undermicrocater and vessel counts were performed. After this the specimenswere processed for histological examination and embedded in wax. The waxblocks were then sectioned at 5 μm and stained with haematoxylin andeosin in a standard fashion. Vessel area density was estimated on allcleared specimens using a microcater which allowed visualizationthroughout the depth of these small tissue specimens. Three fields wererandomly selected at 3 depth intervals of 500 μm and the vessel densitywas assessed with the aid of a stereometric grid. Following completionof this process the specimens were committed to histological processing.The stained sections were morphologically assessed in terms ofangiogenesis and the cellular characteristics of the newly generatedtissue. Univariate analysis of the patency rates and vessel density wasperformed using the Student t-test. The patency rate was assessed forthe two vascular configurations and for the different materials used inthe make-up of the chamber.

[0277] The patency rate for the tied off arteriovenous pedicle was 21%versus 88% for the flow-through pedicle. The patency rate in thepolycarbonate chambers (excluding the tied off AV pedicle group) was 88%versus 97% in the silicone chambers. The new vessels in the tied off AVPgroup were seen to be arising from outside the chamber and growing inalong the thrombosed pedicle. The vessel densities in the flow-throughchambers were similar at 2, 4 and 6 weeks. Similarly there was nodifference in vessel density between PLGA and Matrigel. Morphologicallythere was good angiogenesis in Matrigel® and PLGA but qualitatively itwas better in the Matrigel®. The new vessels seemed to be more numerousand occurred throughout the construct in the Matrigel®. The angiogenesisin the PLGA was more to the periphery of the construct with fewervessels in the central aspect probably due to the solid nature of thisECM.

[0278] In terms of cellular morphology the PLGA seemed to promote apredominately fibrous foreign-body type reaction. Fibroblasts are thepredominant cell s en both peripherally where the matrix lay against thechamber wall and centrally within the substance of the matrix. TheMatrigel® group also showed a fibroblastic response at the ECM-chamberinterface. On the other hand the central aspect of the Matrigel® showsthe presence of fat in the chamber that has clearly migrated through thematrix and survived, presumably nourished by the newly generatedvascular tree. This phenomenon has been reported before innon-encapsulated Matrigel® in mice using growth factors andpre-adipocytes. The presence of mature viable fat in the chambersuggests this model is capable of supporting the migration, maturationand possibly the reproduction of fat cells and their precursors. Infemale animals the fat pad contains some mammary tissue and associatedducts which are occasionally found in the distal part of the chamberwhere this tissue is used as a “plug” to seal the distal aperture. Inthe Matrigel group we observed that in some of these animals the breastductal/acinar tissue seemed to be growing into the Matrigel and inothers there is clear morphological evidence of newly formingductal/acinar tissue. This suggests that the chamber is capable ofsupporting the development of glandular tissue as well as fat. To ourknowledge this has not been reported before.

[0279] We have seeded the chamber with clones of mouse mesenchymal stemcells that were cultured from a C57 Immorto mouse and also with a mousemammary tumour cell line. Both were labelled with flourescent markers(GFP or CFDA) and we were able to demonstrate that the implanted cellswere alive at 48 hours, 4 days, 2 weeks, 4 weeks and 8 weeks. Themammary tumour line has been seen histologically at 4 weeksdemonstrating that the chamber is capable of supporting cell lines inthe longer term. We have also successfully grown foetal pancreas, liver,heart, bowel and limb bud (composite skin, bone, cartilage, muscle,vessel and nerve) in immunodeficient SCID mice. As well as this we havebeen successful in getting cultured adult pancreatic islets to surviveand produce hormones at 2 and 10 weeks in wild type mice (C57BL6). Thiseffectively means that we have successfully grown functioning isletallograft in these animals which has not been achieved in other modelsof pancreatic transplantation. This means that the chamber may confersome immuno-privileged status to the cells that grow within it. This hastherapeutic implications in that it may be possible to use unmatchedallograft or even xenograft in the chamber with or without localimmunosuppression or Sertoli cell co-culture as a treatment of DiabetesMellitus.

[0280] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

[0281] References cited herein are listed on the following pages, andare incorporated herein by this reference.

REFERENCES

[0282] Erol, Ö. O., and Spira, M. Surgery (1980) 66: 109-115.

[0283] Khouri, R. K., Koudsi, B., Deune, E. G., Hong, S. P., Ozbek, M.R., Serdar, C. N., Song, S-Z and Pierce, G. F. Surgery (1993)114:374-380.

[0284] Knight, K. R., Mian, R., Tanaka, Y., Penington, A. J., Hurley, J.V., Cassell, O., Romeo, R., and Morrison, W. A. 7th Annual MeetingAustralian Vascular Biology Society (1999).

[0285] Liu, M., and Shapiro, M. E. Transplant. Proc. (1995) 27:3205-3207.

[0286] Morrison, W. A., Dvir, E., Doi, K., Burley, J. V., Hickey, M. J.,and O'Brien, B. M. Br. J. Plast. Surg. (1990) 43: 645-654.

[0287] Patrick, C. W. Jr., Chauvin, P. B., Hobley, J., and Reece, G. P.Tissue Eng. (1999) 5: 139-151.

[0288] Prockop, D. J. Science (1997) 276: 71-74.

[0289] Tanaka, Y., Tajima, S., Tsutsami, A., Akamatsu, J., and Ohba, S.J. Jpn. P.R.S. (1996) 16: 679-686.

[0290] Tanaka, Y., Tsutsumi, A., Crowe, D. M., Tajima, S., and Morrison,W. A. Br. J. Plast. Surg. (2000) 53: 51-57.

[0291] Vukicevic, S., Kleinman, H. K., Luyten, F. P., Roberts, A. B.,Roche, N. S. and Reddi, A. B. Exp. Cell Res. (1992) 202: 1-8.

[0292] Zuk, P. A., Zhu, M., Mizuno, K., Huang, J., Futrell, W., Katz, A.J., Benhaim, P., Lorenz, H. P., and Hedrick, M. H. Tissue Eng. (2001) 7:211-228.

The claims defining the invention are as follows:
 1. A method ofproducing donor vascularised tissue, suitable for transplantation into arecipient animal in need of such treatment, comprising the steps of: a)creating a functional circulation on a vascular pedicle in a donorsubject; b) partially or totally enclosing the vascular pedicle within afabricated chamber; e) seeding the chamber with isolated cells or piecesof tissue; d) implanting the chamber containing the vascular pedicleinto donor subject at a site where such an anatomical construct can becreated; and e) leaving the chamber in the implantation site for aperiod sufficient to allow the growth of vascularised new tissue.
 2. Amethod according to claim 1, comprising the step of: after step (a)surrounding the vascular pedicle with added extracellular matrix and/ora mechanical support
 3. A method according to claim 1 or claim 2,comprising the step of: after step (b) adding growth factors, drugs,antibodies, inhibitors or other chemicals to the chamber.
 4. A methodaccording to claim 1, in which the vascular pedicle comprises anarterio-venous (AV) loop or shunt.
 5. A method according to claim 1, inwhich the vascular pedicle compris s a ligated artery and vein.
 6. Amethod according to claim 1, wherein the chamber in step (e) is left inthe implantation site for at least 4 weeks.
 7. A method according toclaim 1, wherein the chamber in step (e) is left in the implantationsite for at least 6 weeks.
 8. A method according to claim 1, herein saidcreated vascular pedicle contained within the chamber is connected to anextracorporeal circulation.
 9. A method according to claim 1, whereinthe donor subject of step (a) is a mammal.
 10. A method according toclaim 9, wherein said mammal is a human.
 11. A method according to claim1, comprising the additional step of implanting said vascularised newtissue into an autologous recipient.
 12. A method according to claim 1,comprising the additional step of implanting said vascularised newtissue into a heterologous recipient.
 13. A method according to claim 2,wherein the added extracellular matrix is selected from the groupconsisting of reconstituted basement membrane preparations,polylactic-polyglycolic acid variants (PLGA), fibrin or plasma glue, andnative collagen.
 14. A method according to claim 13, wherein said PLGAcomprises PLGA sponge.
 15. A method according to claim 3, wherein theadditional growth factors, drugs, antibodies, inhibitors or otherchemicals added to the chamber are selected from the group consisting ofgrowth factors, sing factors to attract stem cells from the circulation,exogenous factors and promoters of angiogenesis or vasculogenesis.
 16. Amethod according to claim 1, wherein the isolated cells or pieces oftissue of step (c) are autologous to the host.
 17. A method according toclaim 1, wherein the isolated cells or pieces of tissue of step (c) areheterologous to the host.
 18. A method according to claim 1, wherein theisolated cells or pieces of tissue of step (c) are selected from thegroup consisting of stem cells, skeletal muscle tissue that has beensubjected to ischaemia, myoblasts transfected with Myo-D, keratinocytes,myoblasts, fibroblasts, pre-adipocytes, adipocytes, cardiomyocytes,endothelial cells, smooth muscle cells, chondrocytes, pericytes,bone-marrow derived stromal precursor cells, Schwann cells and othercells of the peripheral and central nervous system, olfactory cells,hepatocytes and other cells of the liver, mesangial cells and othercells of the kidney, pancreatic islet β-cells and ductal cells, thyroidcells, cells of other endocrine organs, portions of skeletal or cardiacmuscle, pancreas, liver, epididymal and other subcutaneous fat, nerves,kidney, bowel, ovary, uterus, testis, and glandular tissue fromendocrine organs.
 19. A method according to claim 1, wherein thefunctional circulation on a vascular pedicle of step (a) comprises anartery and a vein.
 20. A method according to claim 1, wherein thefunctional circulation on a vascular pedicle of step (a) comprises anartery, a venous graft, and a vein.
 21. A method according to clam 1,wherein the functional circulation on a vascular pedicle of step (a)comprises an artery, a venous graft, an arterial graft, and a vein. 22.A method according to claim 1, wherein the functional circulation on avascular pedicle of step (a) comprises the ligated stumps of an arteryand a vein placed side by side.
 23. A vascularised tissue graft producedby a method according to claim
 1. 24. A method of repairing a tissuedeficit, comprising the steps of: a) creating a vascularised tissuegraft according to claim 23; b) retaining the graft in the donor subjectfor a sufficient period to produce tissue with the desired size,vascularity and degree of differentiation; c) transferring the graft tothe desired recipient site; and d) anastomosing the blood vessels of thegraft to a local artery and vein.
 25. A method of tissue augmentation,comprising the steps of: a) creating a vascularised tissue graftaccording to claim 23; b) retaining the graft in the donor subject for asufficient period to produce tissue with the desired size, vascularityand degree of differentiation; c) transferring the graft to the desiredrecipient site; and d) anastomosing the blood vessels of the graft to alocal artery and vein.
 26. A method of delivery of a gene product to asubject, comprising the steps of: a) creating vascularised tissue in atissue chamber according to the method of claim 1; b) removing thechamber with its vascularised tissue and culturing the chamber assemblyin vitro; c) transforming cells of the tissue in the clamber with adesired gene; and d) implanting the vascularised tissue with or withoutthe chamber into a patient in need of such treatment.
 27. A model systemfor vascularised tissue, comprising a tissue chamber comprising anisolated vascular pedicle produced by a method according to claim 1,wherein the tissue chamber is operably connected to an extracorporealcirculation apparatus and to a renal dialysis filter.